Colby College Digital Commons @ Colby Honors eses Student Research 2009 e Effect of Glutamate on Neurite Outgrowth in Fiddler Crab (Uca pugilator) X-organ Cells Ruth B. Langton Colby College Follow this and additional works at: hp://digitalcommons.colby.edu/honorstheses Part of the Biology Commons Colby College theses are protected by copyright. ey may be viewed or downloaded from this site for the purposes of research and scholarship. Reproduction or distribution for commercial purposes is prohibited without wrien permission of the author. is Honors esis (Open Access) is brought to you for free and open access by the Student Research at Digital Commons @ Colby. It has been accepted for inclusion in Honors eses by an authorized administrator of Digital Commons @ Colby. For more information, please contact [email protected]. Recommended Citation Langton, Ruth B., "e Effect of Glutamate on Neurite Outgrowth in Fiddler Crab (Uca pugilator) X-organ Cells" (2009). Honors eses. Paper 470. hp://digitalcommons.colby.edu/honorstheses/470
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Colby CollegeDigital Commons @ Colby
Honors Theses Student Research
2009
The Effect of Glutamate on Neurite Outgrowth inFiddler Crab (Uca pugilator) X-organ CellsRuth B. LangtonColby College
Follow this and additional works at: http://digitalcommons.colby.edu/honorstheses
Part of the Biology CommonsColby College theses are protected by copyright. They may be viewed or downloaded from this sitefor the purposes of research and scholarship. Reproduction or distribution for commercial purposesis prohibited without written permission of the author.
This Honors Thesis (Open Access) is brought to you for free and open access by the Student Research at Digital Commons @ Colby. It has beenaccepted for inclusion in Honors Theses by an authorized administrator of Digital Commons @ Colby. For more information, please [email protected].
Recommended CitationLangton, Ruth B., "The Effect of Glutamate on Neurite Outgrowth in Fiddler Crab (Uca pugilator)X-organ Cells" (2009). Honors Theses. Paper 470.http://digitalcommons.colby.edu/honorstheses/470
Outgrowth in Fiddler Crab (Uca pugilator) X-organ Cells
An Honors Thesis in Biology Ruth B. Langton
Colby College, 2009
The Effect of Glutamate on Neurite Outgrowth in Fiddler Crab (Uca pugilator) X-organ Cells
Ruth Langton
Glutamate is the primary excitatory neurotransmitter in the mammalian central nervous system. It is of particular interest because of its supposed role in the processes of learning and memory, and also because of its potential toxic effects that have been linked to neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. Although glutamate is necessary for normal cell functioning, high levels of glutamate receptor activation can result in cell death, a phenomenon known as excitotoxicity. It has been suggested that glutamate also plays an important role in the insect and crustacean nervous systems, allowing for the examination of excitotoxicity in these organisms. The current study aims to determine the effect of high concentrations of glutamate on the neurite outgrowth of cultured fiddler crab (Uca pugilator) cells. Cells were obtained from the x-organ, a neurohemal organ located in the crustacean eyestalk, and were cultured for 24 hours in simple culture medium. After 24 hours, cells exhibiting neurite outgrowth were photographed and treated with one of four concentrations of glutamate. The treatment groups included: control with 0 mM glu, 0.1 mM glu, 1 mM glu, and 10 mM glu. After another 24 hours, the cells were photographed a second time and the neurite outgrowth was measured and compared. Higher concentrations of glutamate had a negative effect on neurite outgrowth, causing the neurites to retract or slow their growth. Glutamate receptors were also located in the x-organ cells using immunocytochemistry with fluorescence. This study provides insight into the workings of the crustacean nervous system and shows that fiddler crabs are a model organism in which to examine the effects of excitotoxicity, which may lead to future knowledge about the mechanisms of neurodegenerative diseases.
Introduction
Glutamate is the primary excitatory neurotransmitter in the mammalian central
nervous system (Sheldon and Robinson, 2007). It is an amino acid that acts
predominantly on depolarizing post-synaptic receptors (Nicholls, 1993). Compared to all
other neurotransmitters, the levels of glutamate are extremely high in the mammalian
central nervous system, about 1000-fold higher than many other important
neurotransmitters, such as dopamine and serotonin (Sheldon and Robinson, 2007).
Neurotransmitters such as glutamate have been implicated in the development of
neuronal morphology and brain neuroarchitecture, and are important to the overall
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functioning of the nervous system (Mattson, 1988). Glutamate is of particular interest
because of its supposed role in the processes of learning and memory (Bliss and Dolphin,
1982; Collingridge and Singer, 1990) and because of its potential toxic effects that are
associated with neurodegenerative diseases, such as Parkinson’s and Alzheimer’s
(Rothman and Olney, 1986; Choi, 1988; Meldrum and Garthwaite, 1990; Nicholls, 1993;
Sheldon and Robinson, 2007).
It has been shown that excessive activation of glutamate receptors can result in
cell death, a phenomenon known as excitotoxicity (Choi, 1992; Coyle and Puttfarcken,
1993; Doble, 1999; Sheldon and Robinson, 2007). This excitotoxicity has been
associated with a number of acute and chronic neurodegenerative diseases. These
(GluR (H-301)) was added to each dish. Cells were incubated for approximately 1 hour
in the dark at room temperature. Primary antibody was removed and cells were rinsed
with PBS buffer. 200µL of rhodamine phallodin (for actin labeling) and 100µL of goat
anti-rabbit (secondary antibody) were added to each dish and cells incubated of
approximately 30 minutes in the dark at room temperature. Rhodamine and the
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secondary antibody were removed and cells were rinsed with PBS. A few drops of
Prolong Gold antifade fixative with DAPI (for nuclear staining) was added to each dish.
Dishes were stored in an aluminum-wrapped box at room temperature.
Photographing. Cells were located using a Zeiss Axiovert 200 fluorescence microscope.
Photographs were taken using Axiovision software. A confocal microscope at the Mount
Desert Island Biological Laboratory was used to photograph whole chunks of eyestalk
tissue that had been fixed and stained using the procedure above.
Results
Control cells showed more growth after 48 hours than after 24 hours, however,
this difference was not significant (Figure 3, p>0.05). No significant difference among
dishes within each control time period (24 and 28 hours) was found (p>0.05).
Cells treated with 0.1mM glutamate showed a slight but insignificant (p>0.05)
increase after 48 hours of growth when cells were bathed in a 0.1mM glutamate solution
at 24 hours (Figure 4). No significant difference was seen between dishes within each
treatment (p>0.05).
Cells in the 1mM glutamate treatment group also showed a slight but insignificant
(p>0.05) increase from 24 to 48 hours, after being bathed in 1mM glutamate (Figure 5).
No significant difference was seen between culture dishes within each treatment
(p>0.05).
A significant difference was seen between cells in the 10 mM glutamate group
before and after glutamate application (after 24 and 48 hours respectively) (p<0.01). A
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decrease in standardized neurite outgrowth was seen after the application of 10 mM
glutamate (Figure 6). No significant difference was found between culture dishes within
each treatment (p>0.05).
When comparing the four treatment groups, there was no significant difference
seen among any of the groups after 24 hours of growth prior to glutamate application
(Figure 7, p>0.05). After 48 hours (24 hours after the application of glutamate), neurite
outgrowth decreased with increasing concentrations of glutamate, compared to the
control, with 10 mM glutamate cells showing the least outgrowth (Figure 8). The 0.1
mM and 1 mM glutamate treatment groups did not differ significantly from the control
(p>0.05) and are not significantly different from each other (p>0.05). The 10 mM
glutamate treatment group is significantly different from all other treatment groups and
the control (p<0.05).
Immunocytochemistry indicated the potential presence of glutamate receptors in
the fiddler crab neurons using green (FITC) fluorescence. Glutamate receptors appear to
be located on the cell membrane of the cell body and also in the membrane of the neurites
(Figure 9). As well as staining the cultured cells, I also labeled larger pieces of tissue
from the x-organ to determine the general localization pattern of potential glutamate
receptors. Based on the fluorescence staining, the glutamate receptors appear to be
located in the x-organ/sinus gland of the fiddler crab eyestalk (Figure 10), as well as in
other neural tissue.
Cells in the glutamate plus melatonin trial group showed normal neurite
outgrowth after the first untreated 24 hours of growth in simple culture medium. After
being treated with melatonin and glutamate, at 48 hours, the cells had almost all lysed
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and all that was left was debris from the cell bodies. This pattern recurred in all three
different culture dishes.
Discussion
The results of this study suggest glutamate has an effect on neurite outgrowth and
also that glutamate receptors exist in fiddler crab x-organ cells. The lower concentrations
of glutamate (0.1 mM and 1 mM) had no significant impact on neurite outgrowth, but the
highest concentration, 10 mM, significantly decreased outgrowth which is assumed to be
a result of excitotoxicity. I expected that lower levels of glutamate would not affect
normal cell growth, because they are more normal physiological concentrations at which
glutamate is necessary for mammalian cell functioning (Nicholls, 1993). These results
show that high levels of glutamate are necessary to cause excitotoxicity in the x-organ
cells.
Previous studies have also used high concentrations of glutamate and seen a toxic
effect on cells. Ryan et al. (2006) used focal application of glutamate at concentrations
of 5 mM and 25 mM to larval lamprey descending brain neurons. This study found that
the application of glutamate inhibited outgrowth of treated neurites, but did not affect
other neurites from the same neuron (Ryan et al, 2006). However, when glutamate was
applied directly to the cell body, all the neurites from the cell were inhibited (Ryan et al.,
2006), which corresponds with the results of the my study. Owen and Bird (1997) used
concentrations of glutamate ranging from 1 to 100 µM when studying its effects on
mouse spinal cord neurons and found a significant inhibition of neurite growth. This
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suggests that higher concentrations of glutamate are necessary for outgrowth inhibition in
crustaceans than in mammals.
Immunocytochemisty with fluorescence was used in this study to attempt to
locate glutamate receptors, which appear to have been successfully labeled in both the
cell body and the neurites of x-organ cells. This provides support for the inhibitory effect
of glutamate on neurite outgrowth because it indicates that glutamate is able to enter and
interact with the cell. Further studies are needed to more fully establish the presence of
these receptors.
After I determined that 10 mM glutamate significantly decreased neurite
outgrowth in cultured cells, I added melatonin as another variable in the experiment to
determine if melatonin, an antioxidant, has the ability to rescue cells treated with
glutamate. Melatonin is known to have widespread antioxidant actions including the
direct scavenging of free radicals and activation of enzymes in antioxidant pathways (Tan
et al, 1993; Reiter et al., 2007). Antolin, et al. (2002) suggests that melatonin prevents
cell death as well as damage caused by oxidative stress in a mouse model of Parkinson’s
disease and indicate that melatonin may prevent neurotoxin damage in general.
If cells were rescued from the excitotoxicity caused by glutamate, they would not
show reduced outgrowth, or may show more outgrowth after 48 hours. For example,
previous studies in the Tilden lab (Cuttler, 2007) showed both a neurite growth enhancing
effect and a separate antioxidant, neuroprotective effect of melatonin alone on x-organ
cells. However, in a trial run of this experiment, these cells lysed after being treated with
both glutamate and melatonin, and all cells that were present in the dish after 48 hours
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did not have visible neurites. The reason for this detrimental effect of the combination of
melatonin and glutamate is unclear and is an area that requires further research.
In sum, I was able to locate glutamate receptors in the x-organ cells of fiddler
crabs, which implicates these crabs as a powerful model system for studying glutamate-
mediated neurodegeneration. These fiddler crabs can be used in future studies to add to
the body of research about neurodegenerative disease and excitotoxicity.
Acknowledgements
I would like to thank the NIH-INBRE Grant and the Biology Department Honors
Project Funds for providing funding for my project. Thanks to my advisor Dr. Andrea
Tilden for her support and guidance throughout this research project, and my readers Dr.
Cathy Bevier and Dr. Josh Kavaler. I would also like to thank Christina Mok and Adam
Paine for helping to complete experimental trials and gather data. Thanks to Jen Myers
for guiding me through many laboratory procedures.
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Figure 1. Control cell after 48 hours of growth in simple culture medium. Photographed using a Zeiss Axiovert 200 microscope at 400X magnification.
Figure 2. Control cell after 48 hours of growth in simple culture medium. Photographed using a Zeiss Axiovert 200 microscope at 400X magnification and analyzed using AxioVision software. Measurements represent cell body size (269.59µm2) and total neurite encompassing area (2593.29 µm2).
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Figure 3. Mean neurite outgrowth of the control cells, standardized for differing cell body sizes, after 24 and 48 hours of growth in simple culture medium (+/-SE).
Figure 4. Mean neurite growth of cells, standardized for differing cell body sizes, in the 0.1 mM glutamate treatment group after 24 and 48 hours of growth, before and after glutamate application respectively (+/- SE).
Figure 5. Mean neurite growth of cells, standardized for differing cell body sizes, in the 1 mM glutamate treatment group after 24 and 48 hours of growth, before and after glutamate application respectively (+/- SE).
Figure 6. Mean neurite growth of cells, standardized for differing cell body sizes, in the 10 mM glutamate treatment group after 24 and 48 hours of growth, before and after glutamate application respectively (+/- SE).
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Figure 7. Neurite growth, standardized for differing cell body size, after 24 hours and before glutamate application to all treatment groups (+/- SE).
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Figure 8. Neurite growth, standardized for differing cell body size, after 48 hours of growth and after glutamate application to all treatment groups (+/- SE).
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Figure 9. Cluster of six cultured cells labeled with green fluorescence for glutamate receptors (GluR-1, 2, 3, 4). (a) (b)
x-organ/sinus gland area
Figure 10. X-organ/sinus gland area of the fiddler crab eyestalk stained for (a) cell nuclei with DAPI blue fluorescence (b) glutamate receptors (GluR-1, 2, 3, 4) with fitc green fluorescence.